Protocol 35978: Probe Assay - Pop7<em1(IMPC)J>
Version 1.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.

 

The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX). To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility. Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.

Expected Results

>chr5:137502483-137502610 128bp TCAATTAGCCCCGCCTCT GTACTCTCCGCCTCCTTTCC

Mutant=  130 bp

Wild Type = 128 bp

Sequence

Wt sequence deletion in lower case

GAGAACTGGGCGATCCACGCCACGCCCCTCTTGGGCCCGCCTCTGTCAATTAGCCCCGCCTCTGCTAGCTAGTCCCGCCCTTCGAAGCGCTCACTAGAACCCTGCgaaccctcaagtctcgcgagccttaaatcgccttgcgcacgcgcacgaggaaaggaggcggagagtacggaagcctagaaggccataagcttatcggttccggcttggagcggggacgcgcgtgcgcagtgcgtagcccttgctggcgagcctgcccggcgttcctcttccgatcccttctccgttcttcttctgcatatcgaggccagcgacgtccacttggatcggtcggcgaggctcacgcgagtgaagggcactgaggaaggaagaaggtaggcgtggacacgccgggaatgggaaccacgtgatgaatgaattaaaactgggagtggagaaagcggaagtggaagggcttgagacactgggaaggaagagggtgttgagggcgcccccttctggttgcacccttagaaagccgggcaagaaaggattctcctgacctccctgaatcttgttccttctccatgccaggactcagagcatggcagaaaacagagaaccccgctgtgccatcgaggctgagctggaccctgtggagtacacccttcggaagcgacttccccaccgcttgccccggaggcccaacgacatttatgtcaacatgaagactgactttaaggcccagctagctcgctgccaaaagctcctggacggaggcactagggggcagaacgcatgcactgaaatctacattcatggcttgggcctggccatcaaccgggccatcaacatcgccctgcagctgcaggcgggcagctttgggtccctacaggtggccgccaatacctccaccgtggagctggtggacgagttggagccagagactgactctcgggagcccctgacgagggtcaggaacaactcagccatccacatccgagtcttcagggtcacgcccaagtaactgaagggaggctgacttggacccaccaaggcttctgagcacacacatccatgtggccgaggtttctagtgcttcatagtggctcttgttctgagttctgtcctgagcctcttgttaccttgttacttcagcctagaggactgagaatggagagcccgagaagtgaatcttccttgtcctgtataataaattattaagttgtgttggtgccgggcttcgaatggcctctttttcctagtgtaattctgtgtgtacttgaataggaaaatggtaggaaagagcaagtatataggccacagtaaggcatctcacattgaaccacagTATGACGGGAATCAAATTTGTTGAGTGTAACACGGAAGCTTAATTGTTACAGTGTGCTCTCAGGAAGATGAGAGAGTAGACTCATCGACTGAGCT

This is a 1228 bp deletion beginning at Chromosome 5 position 137,501,323 bp and ending after 137,502,550 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
47452 TCA ATT AGC CCC GCC TCT Common A
47453 GTA CTC TCC GCC TCC TTT CC Wild type Reverse A
47454 CAT CTT CCT GAG AGC ACA CTG Mutant Reverse A
47455 Fluorophore-1 AGA ACC CTG CTA TGA CGG G Quencher-1 MUT Probe
47456 Fluorophore-2 TCA AGT CTC GCG AGC CTT Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
47452 0.40 uM
47453 0.40 uM
47454 0.40 uM
Wt Probe 0.15 uM
Mutant Probe 0.15 uM
DNA

Cycling

Step Temp °C Time Note
1 95.0 --
2 95.0 --
3 60.0 --
4 -- repeat steps 2-3 for 40 cycles
5 40.0 -- Forever
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Strains Using This Protocol

This is the only strain that uses this protocol.